1. Field of the Invention
The present invention relates in general to methods and materials for expression of a gene encoding a plasminogen variant and in particular to methods and materials for expression of a human plasminogen variant in a mammalian cell system and products thereof.
2. Description of Related Art
Deleterious accumulations in blood vessels of the clot protein fibrin are prevented by proteolytic degradation (fibrinolysis) of fibrin or of its precursor fibrinogen by the enzyme plasmin (Pm). In a large variety of disorders, pathological fibrin deposits are not degraded spontaneously, resulting in thrombosis, the presence of a blood clot (thrombus) in a blood vessel. In many cases, thrombolytic therapy, i.e., dissolution of the blood clot by Pm, is the only feasible treatment.
Pm is produced in the circulation by activation of a precursor, the "proenzyme" or "zymogen" called plasminogen (Pg). Thrombolytic therapy is conducted by the administration of a plasminogen activator. Among such plasminogen activators are streptokinase (SK), urokinase (UK) and tissue plasminogen activator (t-PA).
Human Pg (HPg) exists in the circulation as a single-chain glycoprotein containing 791 amino acids having an amino-terminal amino acid of Glu (circulating HPg may thus be referred to as [Glu.sup.1 ]plasminogen) [Forsgren et al., FEBS Lett., 213: 254-260 (1987); Malinowski et al., Biochem., 23: 4243-4250 (1984); McLean et al., Nature, 330: 132-137 (1987); Sottrup-Jensen et al., Prog. Chem. Fibrinolysis Thrombolysis, 3: 191-209 (1978); Wiman, Eur. J. Biochem., 39: 1-9 (1973); and Wiman, Eur. J. Biochem., 76: 129-137 (1977)].
Analysis of the carbohydrate sequence of HPg reveals that there are two glycosylation variants, a first having two glycosylation sites (Asn.sup.289 and Thr.sup.346) and a second having one glycosylation site (Thr.sup.346), with subforms exhibiting incomplete sialation [Castellino, Chem. Rev., 81: 431-446 (1981)]. These forms and subforms are examples of post-translational modifications exhibited by circulating plasminogen.
HPg is activated by cleavage of a Arg.sup.561 -Val.sup.562 peptide bond to produce the two-chain, disulfide-linked serine protease [Lys.sup.78 ]Pm. This molecule also lacks the amino-terminal 77 amino acids as a result of autolysis by human Pm (HPm) formed during the activation [(Violand Castellino, J. Biol. Chem., 251: 3906-3912 (1976)]. The cleavage may be catalyzed by a variety of activators, among which are SK, UK and t-PA. [For a review, see Castellino, Bioscience, 33: 647-650 (1983).] Whereas the latter two proteins are enzymes that directly catalyze cleavage of the appropriate peptide bond in HPg, providing HPm, SK has no such inherent activity and its plasminogen activator activity relies on its ability to form complexes with HPg and HPm and utilize the actual or latent plasmin active sites of these latter two molecules to function as an activator [Castellino, supra].
[Glu.sup.1] Pg exists in plasma in the form of two major variants, which differ in their extent of glycosylation at Asn.sup.289 [Hayes and Castellino, J. Biol. Chem., 254: 8768-8780 (1979); Castellino, supra]. In [Glu.sup.1 Pg, the latent plasmin heavy chain, which includes residues 1-561, contains five highly homologous regions called "kringles" [Sottrup-Jensen et al., supra , each containing approximately 80 amino acids. These kringles most likely exist as independent domains [Castellino et al., J. Biol. Chem., 256 4778-4782 (1981)] and are of importance to the functional properties of HPg and HPm. As examples, the kringle 1 domain (amino acid residues 84-162) may be important in the interaction of plasmin or plasminogen with fibrin and fibrinogen [Lucas et al., J. Biol. Chem., 258: 4249-4256 (1983)], with the negative activation effector (Cl.sup.-) [Urano et al., J. Biol. Chem., 262: 15959-15964 (1987)], and with the positive activation effector epsilon-aminocaproic acid (EACA) [Markus et al., J. Biol. Chem., 253: 727-732 (1978)]. Additionally, this same segment is responsible for the initial rapid binding of HPm to its major plasma inhibitor, .alpha..sub.2 -antiplasmin [Moroi and Aoki, J. Biol. Chem., 251: 5956-5965 (1976)]. The kringle 4 region (residues 358-435 ) appears to contain weak EACA binding site(s) present on [Glu.sup.1 ]Pg, which may be involved in the very large ligand-induced conformational alteration of [Glu.sup.1 Pg [Violand et al., Arch. Biochem. Biophys., 170: 300-305 (1975)] and in a concomitant increase in the activation rate of the zymogen in the presence of the positive effector EACA [Claeys and Vermyelin, Biochem. Biophys. Acta, 342, 351-359 (1974)].
Although thrombolytic therapy is useful, its therapeutic potential is constrained by the availability of plasminogen at the site of the thrombus. The concentration of plasminogen may be limited due to consumption of plasminogen as a result of thrombolytic therapy, due to an inadequate amount of plasminogen being present in thrombi, or to a local plasminogen depletion related to the age of the thrombus and ischemia (a localized anemia due to a reduction in blood flow). [Anderle et al., Haemostasis, 18: (Suppl. 1), 165-175 (1988)]. Thus, supplementation of the locally available amount of plasminogen is desirable.
Although expression of large amounts of plasminogen in a recombinant expression system is a convenient way to obtain plasminogen for use in thrombolytic therapy, there have been great difficulties in expression of intact HPg in mammalian expression systems due to the nearly ubiquitous presence of intracellular plasminogen activators among mammalian cell types. The presence of these activators results in the appearance of a degraded form of HPg in conditioned cell media of such expression systems, possibly form autodigestion of plasminogen by the HPm produced [Busby et al., Fibrinolysis, 2, 64 (1988)].
A recombinant human plasminogen has been produced in insect cells (irHPg) that, by amino-terminal amino acid sequence analysis, molecular weight estimation on SDS/PAGE, Sepharoselysine affinity chromatography behavior, activation characteristics, antibody reactivity, and activity of the resulting plasmin, appears to be comparable in properties to human plasma [Glu.sup.1 ]Pg. [Whitefleet-Smith et al., Arch. Biochem. Biophys., 271: 390-399 (1989)]. This is a significant finding, since to date there has not been successful expression of wild-type recombinant HPg (wt-rHPg) in mammalian cells. However, when the kinetic properties of equimolar complexes formed from streptokinase with plasma HPg and with irHPg were compared, SDS/PAGE gels of the temporal events within the respective complexes were identical to results published by Bajaj and Castellino, J. Biol. Chem., 252: 492-498 (1977), in that a rapid conversion of HPg to HPm occurred. This suggests that HPg is not a stable component of the complex.
It is known that the cleavage site of human t-PA encompassing positions 270-280 can be modified to create a t-PA variant that is resistant or immune to specific enzymatic cleavage. For example, t-PA variants are described [EP Pat. Publ. No. 199,574] that have amino acid substitutions at the proteolytic cleavage sites at positions 275, 276, and 277. These variants, characterized preferentially as t-PA variants having an amino acid other than arginine at position 275, are referred to as protease-resistant one-chain t-PA variants; unlike natural t-PA, which can exist in either a one-chain or two-chain form, they are resistant to protease cleavage at position 275 and are therefore not converted metabolically in vivo into a two-chain form. This form of t-PA is thought to have certain advantages biologically and commercially, in that it is more stable and its fibrin binding and fibrin stimulation are increased relative to two-chain t-PA. Another form of plasminogen activator contains one domain capable of interacting with fibrin and the protease domain of urokinase, with one embodiment being urokinase altered to make it less susceptible to forming two-chain urokinase. See WO 88/05081 published Jul. 14, 1988.
For further patent literature regarding modification of the protease cleavage site of t-PA, see, for example, EPO Pat. Nos. 241,209; 201,153 published Nov. 12, 1986; 233,013 published Aug. 19, 1987; 292,009 published Nov. 23, 1988; 293,936 published Dec. 7, 1988; and 293,934 published Dec. 7, 1988; and WO 88/10119.
It is an object of the present invention to devise a plasminogen molecule that is stable as such in a complex with a fibrinolytic enzyme such as streptokinase and is hence more active than the natural molecule.
It is another object to produce the plasminogen molecule in any recombinant expression system, not just those that lack an endogenous, site-specific plasminogen activator (such as insect cells).
These and other objects will become apparent to one of ordinary skill in the art.